The MAT1 locus is required for microconidia-mediated sexual fertility in the rice blast fungus.

Rice blast fungus (Pyricularia oryzae) is a heterothallic ascomycete that causes the most destructive disease in cultivated rice worldwide. This fungus reproduces sexually and asexually, and its mating type is determined by the MAT1 locus, MAT1-1 or MAT1-2. Interestingly, most rice-infecting field isolates show a loss of female fertility, but the MAT1 locus is highly conserved in female-sterile isolates. In this study, we performed a functional analysis of MAT1 using the CRISPR/Cas9 system in female- and male-fertile isolates and female-sterile (male-fertile) isolates. Consistent with a previous report, MAT1 was essential for sexual reproduction but not for asexual reproduction. Meanwhile, deletion mutants of MAT1-1-1, MAT1-1-2, and MAT1-1-3 exhibited phenotypes different from those of other previously described isolates, suggesting that the function of MAT1-1 genes and/or their target genes in sexual reproduction differs among strains or isolates. The MAT1 genes, excluding MAT1-2-6, retained their functions even in female-sterile isolates, and deletion mutants lead to loss or reduction of male fertility. Although MAT1 deletion did not affect microconidia (spermatia) production, microconidia derived from the mutants could not induce perithecia formation. These results indicated that MAT1 is required for microconidia-mediated male fertility in addition to female fertility in P. oryzae .

Overexpression of Rice BSR2 Confers Disease Resistance and Induces Enlarged Flowers in Torenia fournieri Lind.

Plant pathogens evade basal defense systems and attack different organs and tissues of plants. Genetic engineering of plants with genes that confer resistance against pathogens is very effective in pathogen control. Conventional breeding for disease resistance in ornamental crops is difficult and lagging relative to that in non-ornamental crops due to an inadequate number of disease-resistant genes. Therefore, genetic engineering of these plants with defense-conferring genes is a practical approach. We used rice BSR2 encoding CYP78A15 for developing transgenic Torenia fournieri Lind. lines. The overexpression of BSR2 conferred resistance against two devastating fungal pathogens, Rhizoctonia solani and Botrytis cinerea. In addition, BSR2 overexpression resulted in enlarged flowers with enlarged floral organs. Histological observation of the petal cells suggested that the enlargement in the floral organs could be due to the elongation and expansion of the cells. Therefore, the overexpression of BSR2 confers broad-spectrum disease resistance and induces the production of enlarged flowers simultaneously. Therefore, this could be an effective strategy for developing ornamental crops that are disease-resistant and economically more valuable.

OsNAC111, a blast disease-responsive transcription factor in rice, positively regulates the expression of defense-related genes.

Plants respond to pathogen attack by transcriptionally regulating defense-related genes via various types of transcription factors. We identified a transcription factor in rice, OsNAC111, belonging to the TERN subgroup of the NAC family that was transcriptionally upregulated after rice blast fungus (Magnaporthe oryzae) inoculation. OsNAC111 was localized in the nucleus of rice cells and had transcriptional activation activity in yeast and rice cells. Transgenic rice plants overexpressing OsNAC111 showed increased resistance to the rice blast fungus. In OsNAC111-overexpressing plants, the expression of several defense-related genes, including pathogenesis-related (PR) genes, was constitutively high compared with the control. These genes all showed blast disease-responsive expression in leaves. Among them, two chitinase genes and one ß-1,3-glucanase gene showed reduced expression in transgenic rice plants in which OsNAC111 function was suppressed by a chimeric repressor (OsNAC111-SRDX). OsNAC111 activated transcription from the promoters of the chitinase and ß-1,3-glucanase genes in rice cells. In addition, brown pigmentation at the infection sites, a defense response of rice cells to the blast fungus, was lowered in OsNAC111-SRDX plants at the early infection stage. These results indicate that OsNAC111 positively regulates the expression of a specific set of PR genes in the disease response and contributes to disease resistance.

WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance.

OsWRKY76 encodes a group IIa WRKY transcription factor of rice. The expression of OsWRKY76 was induced within 48h after inoculation with rice blast fungus (Magnaporthe oryzae), and by wounding, low temperature, benzothiadiazole, and abscisic acid. Green fluorescent protein-fused OsWRKY76 localized to the nuclei in rice epidermal cells. OsWRKY76 showed sequence-specific DNA binding to the W-box element in vitro and exhibited W-box-mediated transcriptional repressor activity in cultured rice cells. Overexpression of OsWRKY76 in rice plants resulted in drastically increased susceptibility to M. oryzae, but improved tolerance to cold stress. Microarray analysis revealed that overexpression of OsWRKY76 suppresses the induction of a specific set of PR genes and of genes involved in phytoalexin synthesis after inoculation with blast fungus, consistent with the observation that the levels of phytoalexins in the transgenic rice plants remained significantly lower than those in non-transformed control plants. Furthermore, overexpression of OsWRKY76 led to the increased expression of abiotic stress-associated genes such as peroxidase and lipid metabolism genes. These results strongly suggest that OsWRKY76 plays dual and opposing roles in blast disease resistance and cold tolerance.

Identification of natural diterpenes that inhibit bacterial wilt disease in tobacco, tomato and Arabidopsis.

The soil-borne bacterial pathogen Ralstonia solanacearum invades a broad range of plants through their roots, resulting in wilting of the plant, but no effective protection against this disease has been developed. Two bacterial wilt disease-inhibiting compounds were biochemically isolated from tobacco and identified as sclareol and cis-abienol, labdane-type diterpenes. When exogenously applied to their roots, sclareol and cis-abienol inhibited wilt disease in tobacco, tomato and Arabidopsis plants without exhibiting any antibacterial activity. Microarray analysis identified many sclareol-responsive genes in Arabidopsis roots, including genes encoding or with a role in ATP-binding cassette (ABC) transporters, and biosynthesis and signaling of defense-related molecules and mitogen-activated protein kinase (MAPK) cascade components. Inhibition of wilt disease by sclareol was attenuated in Arabidopsis mutants defective in the ABC transporter AtPDR12, the MAPK MPK3, and ethylene and abscisic acid signaling pathways, and also in transgenic tobacco plants with reduced expression of NtPDR1, a tobacco homolog of AtPDR12. These results suggest that multiple host factors are involved in the inhibition of bacterial wilt disease by sclareol-related compounds.

Rice WRKY45 plays important roles in fungal and bacterial disease resistance.

Plant 'activators', such as benzothiadiazole (BTH), protect plants from various diseases by priming the plant salicylic acid (SA) signalling pathway. We have reported previously that a transcription factor identified in rice, WRKY45 (OsWRKY45), plays a pivotal role in BTH-induced disease resistance by mediating SA signalling. Here, we report further functional characterization of WRKY45. Different plant activators vary in their action points, either downstream (BTH and tiadinil) or upstream (probenazole) of SA. Rice resistance to Magnaporthe grisea, induced by both types of plant activator, was markedly reduced in WRKY45-knockdown (WRKY45-kd) rice, indicating a universal role for WRKY45 in chemical-induced resistance. Fungal invasion into rice cells was blocked at most attempted invasion sites (pre-invasive defence) in WRKY45-overexpressing (WRKY45-ox) rice. Hydrogen peroxide accumulated within the cell wall underneath invading fungus appressoria or between the cell wall and the cytoplasm, implying a possible role for H(2)O(2) in pre-invasive defence. Moreover, a hypersensitive reaction-like reaction was observed in rice cells, in which fungal growth was inhibited after invasion (post-invasive defence). The two levels of defence mechanism appear to correspond to Type I and II nonhost resistances. The leaf blast resistance of WRKY45-ox rice plants was much higher than that of other known blast-resistant varieties. WRKY45-ox plants also showed strong panicle blast resistance. BTH-induced resistance to Xanthomonas oryzae pv. oryzae was compromised in WRKY45-kd rice, whereas WRKY45-ox plants were highly resistant to this pathogen. However, WRKY45-ox plants were susceptible to Rhizoctonia solani. These results indicate the versatility and limitations of the application of this gene.

Enhanced soybean infection by the legume "super-virulent" Agrobacterium tumefaciens strain KAT23.

Agrobacterium tumefaciens KAT23 harbors a nopaline-type Ti plasmid and is "super-virulent" to soybean (Glycine max) and other leguminous plants. The right and left border sequences of the essential cis-element for T-DNA transfer were removed in order to utilize the high infectivity of this strain in an Agrobacterium-mediated soybean transformation system. The resulting strain, named Soy2, showed no oncogenic activity. After inoculation with disarmed Soy2 harboring binary vector pIG121-Hm and pCAMBIA-WR, soybean epicotyls exhibited high beta-glucuronidase activities, with efficiencies higher than EHA105, an A. tumefaciens strain widely used in making transgenic plants.

Characterization and host range determination of soybean super virulent Agrobacterium tumefaciens KAT23.

Agrobacterium tumefaciens KAT23 isolated from peach root causes crown gall disease in a number of grain legume plants, including the common bean (Phaseolus vulgaris) and soybean (Glycine max). KAT23 caused tumor formation in each of these plants more effectively than strain C58. Biotype determination suggested that this strain is biotype II. KAT23 was able to utilize nopaline as a carbon source. Partial sequence analysis indicated that KAT23 harbors a nopaline-type Ti plasmid, designated pTiKAT23, which was highly homologous with other nopaline-type Ti plasmids (pTiC58 and pTiSAKURA). KAT23 transferred not only the T-DNA of the Ti plasmid but also introduced T-DNA of the binary vector efficiently. The common bean inoculated with KAT23 (pIGFP121-Hm) showed crown galls, and some plants showed beta-glucuronidase (GUS) and sGFP (S65T) gene expression. This virulent ability of KAT23 indicates its potential application to legumes, especially to soybean transformation.

gid1, a gibberellin-insensitive dwarf mutant, shows altered regulation of probenazole-inducible protein (PBZ1) in response to cold stress and pathogen attack.

A recessive gibberellin (GA)-insensitive dwarf mutant of rice, gibberellin-insensitive dwarf1 (gid1), has been identified, which shows a severe dwarf phenotype and contains high concentrations of endogenous GA. To elucidate the function of gid1, proteins regulated downstream of gid1 were analysed using a proteomic approach. Proteins extracted from suspension-cultured cells of gid1 and its wild type were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Of a total of 962 proteins identified from the suspension-cultured cells, 16 were increased and 14 were decreased in gid1 compared with its wild type. Among the proteins hyper-accumulated in gid1 were osmotin, triosephosphate isomerase, probenazole inducible protein (PBZ1) and pathogenesis-related protein 10. Of these four genes, only the expression of PBZ1 was increased by exogenous GA3 application. Expression of this gene was also enhanced in shoots of the wild type by cold stress or by rice blast fungus infection. Under normal growth conditions, there was more PBZ1 protein in gid1 than in the wild type. In addition, gid1 showed increased tolerance to cold stress and resistance to blast fungus infection. The entcopalyl diphosphate synthase (OsCPS) genes, which encode enzymes at the branch point between GA and phytoalexin biosynthesis, were expressed differentially in gid1 relative to the wild type. Specifically, OsCPS1, which encodes an enzyme in the GA biosynthesis pathway, was down-regulated and OsCPS2 and OsCPS4, which encode enzymes in phytoalexin biosynthesis, were up-regulated in gid1. These results suggest that the expression of PBZ1 is regulated by GA signalling and stress stimuli, and that gid1 is involved in tolerance to cold stress and resistance to blast fungus.

Identification of novel HrpXo regulons preceded by two cis-acting elements, a plant-inducible promoter box and a -10 box-like sequence, from the genome database of Xanthomonas oryzae pv. oryzae.

A regulatory protein HrpXo of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, is known to control the expression of hrp genes that encode components of a type III secretion system and of some effector protein genes. In this study, we screened novel HrpXo regulons from the genome database of X. oryzae pv. oryzae, searching for ORFs preceded by two predicted sequence motifs, a plant-inducible promoter box-like sequence and a -10 box-like sequence. Using a gus reporter system, nine of 15 ORF candidates were expressed HrpXo dependently. We also showed by base-substituted mutagenesis that both motifs are essential for the expression of the genes.

Gene involved in transcriptional activation of the hrp regulatory gene hrpG in Xanthomonas oryzae pv. oryzae.

A novel regulatory gene, trh, which is involved in hrp gene expression, is identified in the plant pathogen Xanthomonas oryzae pv. oryzae. In the trh mutant, expression of HrpG, which is a key regulator for hrp gene expression, is reduced both under the in vitro hrp-inducing condition and in planta.

A proteomic approach to analyze auxin- and zinc-responsive protein in rice.

Auxin and zinc are involved in callus and root formation in rice. However, details of the mechanism underlying this process and functional relation between zinc and auxin are unclear. In this study, proteins induced by auxin and zinc in rice were analyzed by a proteomic approach. Root formation on rice seedlings was promoted by 0.45 microM 2,4-D treatment and was further promoted by addition of 260 microM Zn. Microscopic observation revealed that the number of root primodia formed was significantly increased in 2,4-D- and Zn-treated seedlings than that of the control. A total of seven proteins, as analyzed by 2D-PAGE, were increased, and one protein was decreased by 2,4-D and Zn treatment. Expression of elongation factor-1beta' (EF-1beta') both at transcriptional and translational levels was particular abundant in callus and basal parts of young seedlings, and the accumulation of EF-1beta' was consistent with root formation induced by 2,4-D and Zn. Results indicate that higher level of EF-1beta' expression is necessary for auxin- and zinc-induced root formation in rice.

Effects on promoter activity of base substitutions in the cis-acting regulatory element of HrpXo regulons in Xanthomonas oryzae pv. oryzae.

In Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, HrpXo is known to be a transcriptional regulator for the hypersensitive response and pathogenicity (hrp) genes. Several HrpXo regulons are preceded by a consensus sequence (TTCGC-N(15)-TTCGC), called the plant-inducible promoter (PIP) box, which is required for expression of the gene that follows. Thus, the PIP box can be an effective marker for screening HrpXo regulons from the genome database. It is not known, however, whether mutations in the PIP box cause a complete loss of promoter activity. In this study, we introduced base substitutions at each of the consensus nucleotides in the PIP box of the hrpC operon in X. oryzae pv. oryzae, and the promoter activity was examined by using a beta-glucuronidase (GUS) reporter gene. Although the GUS activity was generally reduced by base substitutions, several mutated PIP boxes conferred considerable promoter activity. In several cases, even imperfect PIP boxes with two base substitutions retained 20% of the promoter activity found in the nonsubstituted PIP box. We screened HrpXo regulon candidates with an imperfect PIP box obtained from the genome database of X. oryzae pv. oryzae and found that at least two genes preceded by an imperfect PIP box with two base substitutions were actually expressed in an HrpXo-dependent manner. These results indicate that a base substitution in the PIP box is quite permissible for HrpXo-dependent expression and suggest that X. oryzae pv. oryzae may possess more HrpXo regulons than expected.

Evidence for HrpXo-dependent expression of type II secretory proteins in Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae is a causal agent of bacterial leaf blight of rice. Recently, an efficient hrp-inducing medium, XOM2, was established for this bacterium. In this medium, more than 10 proteins were secreted from the wild-type strain of X. oryzae pv. oryzae. Many of these proteins disappeared or decreased in amount in culture on XOM2 when incubated with the strain that has a mutation in the hrp regulatory gene. Interestingly, the secretory protein profile of a mutant lacking a type III secretion system (TTSS), components of which are encoded by hrp genes, was similar to that of the wild-type strain except that a few proteins had disappeared. This finding suggests that many HrpXo-dependent secretory proteins are secreted via systems other than the TTSS. By isolating mutant strains lacking a type II secretion system, we examined this hypothesis. As expected, many of the HrpXo-dependent secretory proteins disappeared or decreased when the mutant was cultured in XOM2. By determining the N-terminal amino acid sequence, we identified one of the type II secretory proteins as a cysteine protease homolog, CysP2. Nucleotide sequence analysis revealed that cysP2 has an imperfect plant-inducible-promoter box, a consensus sequence which HrpXo regulons possess in the promoter region, and a deduced signal peptide sequence at the N terminus. By reverse transcription-PCR analysis and examination of the expression of CysP2 by using a plasmid harboring a cysP2::gus fusion gene, HrpXo-dependent expression of CysP2 was confirmed. Here, we reveal that the hrp regulatory gene hrpXo is also involved in the expression of not only hrp genes and type III secretory proteins but also some type II secretory proteins.

Histopathology of Red Stripe of Rice.

A histological study of red stripe of rice was conducted to elucidate the mode of infection of the causal bacterium Microbacterium sp. When pin-point-sized spots first appeared at 3 days after inoculation, the bacterial cells had entered through stomata and multiplied in the intercellular spaces of substomatal parenchymatous tissues. With the early appearance of small yellow spots at 4 to 5 days after inoculation, the bacterium was detected in some xylem vessels as well as in parenchymatous tissues, and it had apparently translocated directly from parenchymatous tissues to transverse vascular systems through spiral vessel walls. With the appearance of typical red stripe symptoms comprised of orange lesions and halos at 8 days after inoculation, bacterial masses were present in transverse and longitudinal vascular bundles in areas with orange lesions. In the areas with orange to light brown spots, granules that stained dark blue using Stoughton's method appeared in the protoplasm of the host parenchymatous cells, which later became necrotic. In halo areas, bacterial masses were observed only in some cases, and chloroplasts were disorganized. Bacterial infection was also confirmed by observing sections of naturally infected samples, and the distribution of bacteria was much more extensive than in artificially inoculated samples.

Involvement of Phosphoglucose Isomerase in Pathogenicity of Xanthomonas oryzae pv. oryzae.

ABSTRACT Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, was subjected to transposon mutagenesis to generate mutants defective in pathogenicity. A novel mutant 74M913 was attenuated in virulence but retained its ability to cause the hypersensitive response in leaf blight-resistant rice and tomato. Cloning and sequence analysis revealed that the transposon in 74M913 was inserted in a gene homologous to the phosphoglucose isomerase (pgi) gene of X. axonopodis pv. citri. Growth of the mutant in a synthetic medium containing fructose or xylose as a sole carbohydrate source was much reduced, indicating the transposon disrupted pgi function. The interaction between expression of pgi and hypersensitive response and pathogenicity (hrp) genes was investigated because we had demonstrated previously that expression of hrp genes of X. oryzae pv. oryzae is induced in a synthetic medium containing xylose. However, pgi and the hrp gene (hrcU) were expressed independently. This study suggests that PGI is involved in pathogenicity of X. oryzae pv. oryzae.

Transcriptional response of soybean suspension-cultured cells induced by Nod factors obtained from Bradyrhizobium japonicum USDA110.

Genes responding to Nod factors were picked up by the application of a differential display method for soybean suspension-cultured cells. Forty-five cDNA fragments derived from such genes were detected. Seven fragments (ssc1-ssc7) were successfully cloned. The putative product of genes corresponding to ssc1 was estimated to be a disease-resistance protein relating to the induction of the plant defense response against pathogens, and that corresponding to ssc7 was a sucrose transporter. Amino acid sequences deduced from full-length cDNA corresponding to ssc2 and ssc4 were investigated, and it was shown that these polypeptides were equipped with a leucine zipper motif and with phosphorylation sites that were targeted by tyrosin kinase and cAMP-dependent protein kinase, respectively. In a differential display experiment, the transcriptional levels of three genes corresponding to ssc2, ssc3 and ssc5 were estimated to be up-regulated at 6 h after initiation of the treatment and the remaining four were estimated to be down-regulated. However, transcription of the genes corresponding to all ssc was clearly repressed within 2 h after initiation of the treatment. Five of them were restored to their transcriptional level 6 h after initiation of the treatment, although the others were repressed throughout the experimental period.